Generation of a CDR H3 Randomized Monoclonal Antibody Library by Kunkel Mutagenesis

Complementary-determining regions (CDRs) are the primary factor that determines antibody diversity, while CDR H3 plays a central role in antigen binding. Randomization of this loop within a stable framework provides a powerful strategy for generating synthetic antibody libraries. This chapter outlines an approach that is Kunkel mutagenesis based, for constructing a monoclonal antibody library diversified at CDR H3. The method uses uracilated single-stranded DNA templates and degenerate oligonucleotides to introduce targeted variability while suppressing nonrecombinant background through strategically placed stop codons and restriction sites. The resulting repertoire is directly compatible with phage display for downstream selection. This streamlined protocol enables the generation of antibody libraries suitable for applications in affinity maturation, binder discovery, and antibody development.